The long-term goal of this project is to define the role of plasmid- mediated genes in the pathogenesis of systemic non-typhoid Salmonella infections. The entire 8.2 kb essential virulence region of the S. dublin plasmid pSDL2 has been sequenced, and mutation analysis has defined required loci within the sequence, now termed the spv genes, conserved in all virulence plasmids. The spv genes are induced in response to growth limitation in vitro, and expression is dependent on the chromosomal starvation regulatory locus katF (rpoS), leading to the hypothesis that the plasmid virulence locus is involved in the adaptation of the organism to growth limitation by the host, most likely in the intracellular environment of the macrophage. In this proposal, essential structural genes of the plasmid virulence locus will be defined by site-specific mutations. The molecular mechanism for the regulation of virulence gene expression will be elucidated by a combined biochemical and genetic approach. The site of action of the spv genes in vivo will be determined in the spleen of infected mice, and an in vitro cell culture system will be developed to reflect the activity of the spv locus in vivo. The importance and role of each spv gene will be identified. The following Specific Aims are proposed: l) to define the role of each spv gene in the virulence encoded by the wild-type plasmid in S. dublin. Non-polar spv mutations in the complete virulence plasmid will be constructed and tested in the mouse model of salmonellosis. 2) to define the molecular mechanism for transcriptional activation of the spv operon by the SpvR regulatory protein, using a combination of in vitro DNA binding studies and in vivo genetic analysis. 3) to determine the molecular mechanism for regulation of the spv genes by the chromosomal starvation-induced alternative sigma- factor KatF (RpoS). 4) to establish whether plasmid-mediated proliferation of S. dublin within the spleen takes place within macrophages. Infected spleens will be harvested, the cells dispersed and sorted by FACS into different populations, and the number of viable bacteria per cell will be determined. 5) to establish an in vitro cell culture system using splenic macrophages to examine the role of the spv genes on intracellular growth in resting and cytokine-activated macrophages.